1. Field of Invention
The invention relates to methods of sequencing or assaying nucleotides in solution using antisense probes having their backbones modified to alter their binding characteristics. In particular, the invention relates to sequencing or assaying nucleotides in solution using partially anionic antisense probes, which are not as anionic as unmodified DNA/RNA (i.e., purely phosphodiester) probes, or using nonionic antisense probes.
2. Description of Related Art
DNA/RNA analogs having modified backbones have been employed in a variety of contexts, including therapeutics and diagnostics. In the therapeutic context, modifications of oligonucleotides have largely been motivated by a desire to enhance nuclease resistance and thus increase the longevity of oligonucleotides in vivo. These modifications have generally taken place on the phosphorus atom of the sugar-phosphate backbone, converting the native phosphodiester backbone to other forms. Phosphorothioates, methyl phosphonates, phosphoramidates and phosphotriesters have been reported to confer various levels of nuclease resistance; however, it has been reported that the phosphate modified oligonucleotides have generally suffered from inferior hybridization properties. See, e.g., Cohen, J. S., ed. Oligonucleotides: Antisense Inhibitors of Gene Expression, (CRC Press, Inc., Boca Raton Fla., 1989) and U.S. Pat. No. 5,610,289 to Cook et al.
Despite such reports of inferior hybridization properties, it has been theorized that hybridization efficiency could be improved by eliminating the negative charge on oligonucleotide probes (or going a step further to provide positively charged probes), to thereby eliminate the unfavorable electronic interaction between the probe and the negatively charged target. Thus, much of the DNA/RNA analog art has focused on hybridization employing non-ionic and cationic analogs. See, e.g., U.S. Pat. Nos. 5,677,437 to Teng et al., 5,623,065 to Cook et al., 5,618,704 to Sanghvi et al., 5,602,240 to De Mesmaeker et al., 5,587,469 to Cook et al., 5,541,307 to Cook et al., 5,521,063 to Summerton et al., 5,470,974 to Summerton et al., 5,405,938 to Summerton et al., 5,223,618 to Cook et al., 5,166,330 to Engels et al., 5,166,315 to Summerton et al., 5,142,047 to Summerton et al. and 4,469,863 to Ts'o et al.
A peptide nucleic acid (PNA) is an example of a non-ionic DNA/RNA analog. See, e.g., U.S. Pat. No. 5,539,082 to Nielsen et al. Antisense probes comprising PNA sequences have been employed to detect target nucleotide sequences. For example, U.S. Pat. No. 5,503,980 to Cantor suggests employing PNA probes in a method of sequencing a nucleic acid by hybridizing the nucleic acid with a set of PNA probes containing random, but determinable, base sequences within the single stranded portion adjacent to a double stranded portion, wherein the single stranded portion of the set preferably comprises every possible combination of sequences over a predetermined range. Hybridization occurs by complementary recognition of the single stranded portion of a target with the single stranded portion of the probe and is thermodynamically favored by the presence of adjacent double strandedness of the probe.
However, although Cantor discloses that the nucleic acids can be PNAs, it does not disclose or suggest utilizing such probes in the absence of a solid support. Moreover, the present invention does not require the adjacent construct of DNA material being tested.
In addition to teaching the use of a solid support like Cantor, Perry-O'Keefe et al., "Peptide Nucleic Acid Pre-Gel Hybridization: An Alternative to Southern Hybridization," 93 Proc. Natl. Acad. Sci. USA 14670 (December 1996) also teaches that PNA does not generally bind well to double stranded DNA (dsDNA). See Perry-O'Keefe et al. at page 14673, footnote. Moreover, the homopyrimidine PNA constructs which have been found to bind dsDNA well would not be useful as probes. Applicants have discovered that the qualification which suggests that only homopyrimidine can bind with dsDNA by strand invasion is incorrect and arises from the hybridization conditions employed. Smulevitch et al., "Enhancement of Strand Invasion by Oligonucleotides Through Manipulation of Backbone Charge," 14 Nature Biotechnology 1700 (December 1996) (disclosed in Landsdorp, "Close Encounters of the PNA Kind," 14 Nature Biotechnology 1653 (December 1996)) discloses using PNA primers to hybridize with dsDNA. However, Smulevitch et al. teaches the use of gels in detecting hybridization, and does not suggest the use of fluorescent markers.
Many types of sample analysis rely upon the fluorescent properties of a marker. Fluorescence occurs when a molecule excited by light of one wavelength returns to the unexcited (ground) state by emitting light of a longer wavelength. The exciting and emitted light, being of different wavelengths, can be separated from one another using optical filters, a camera or a CCD. Fluorescence has been used to visualize certain molecules (and hence structures) by light microscopy for many years, and is also used in other analytical techniques, such as flow cytometry. Further, the emission of fluorescence showing different colors can be detected by a human eye, a camera, a charge coupled device (CCD) or a photomultiplier.
For example, U.S. Pat. No. 5,594,138 to Dykstra et al. discloses a method of fluorescent detection of a nucleic acid. The method comprises contacting the nucleic acid with a fluorescent marker that is a bis-dicationic aryl furan compound and exposing the nucleic acid to light at a frequency inducing fluorescence of the fluorescent marker. The fluorescent marker may be conjugated to a nucleotide sequence as a probe for hybridization studies, or it may be conjugated to numerous reagents for in situ labeling studies. Hybridization occurs on a solid support.
U.S. Pat. No. 4,963,477 to Tchen discloses a probe of high sensitivity containing a modified nucleic acid, which can be recognized by specific antibodies.
Fluorescent In Situ Hybridization (FISH) is a technique comprising detecting fluorescent probe binding to human chromosomes by attaching DNA to a solid support, such as a glass slide. See, e.g., K. H. Andy Choo, Ed., "In Situ Hybridization Protocols," Chapters 2 and 4 (Humana Press, Totowa, N.J., 1994). Like all other conventional detection methods comprising hybridization with probes, this method relies on the solid support to keep the two complementary strands of DNA apart while the probe hybridizes with one of the strands. In addition, FISH requires a complicated buffer and temperature control protocol, with overnight incubation.
U.S. Pat. Nos. 5,538,848 to Livak et al. and 4,220,450 to Maggio disclose fluorescence-based detection of nucleotide sequences using oligonucleotide probes in solution; however, these patents require the use of a quenching agent in combination with a reporting agent, so as to distinguish between the signals generated by hybridized probes and unhybridized probes. Livak et al. also requires the use of enzymes in its disclosed method. Quenching agents and enzymes add complexity and expense to the methods.
U.S. Pat. No. 5,332,659 to Kidwell discloses a method for detecting nucleotide sequences in solution using probes comprising at least two fluorophore moieties. The fluorophores must be selected to electronically interact with each other when close enough to vary the wavelength dependence of their spectra. Unhybridized probes are much more flexible than probes hybridized to the target sequence, and consequently the two fluorophore moieties on each probe are more likely to be close to each other when the probe is unhybridized than when the probe is hybridized. Thus, a change in emission wavelength correlated with free probe can be monitored as an indication of the amount of free probe in the sample.
U.S. Pat. No. 5,674,698 to Zarling et al. discloses fluorescent assaying methods comprising the use of "up-converting" labels, which fluoresce at frequencies higher than their excitation frequencies and at wavelengths lower than their excitation wavelengths. Zarling et al. strongly teaches away from assays using down-converting labels (i.e., labels that fluoresce at frequencies lower than their excitation frequencies and at wavelengths higher than their excitation wavelengths) due to poor signal-to-noise ratios.
Until the present invention, however, it has not been possible to rapidly test for the presence of nucleotide sequences in solution using a method which does not destroy the sample, is less hazardous to laboratory personnel than radiation based assays, does not require the cost and delay of separating unhybridized probes from hybridization complexes, does not require the provision of quenching agents, does not require the provision of enzymes, does not require the provision of multiple interactive reporting moieties on, or in the vicinity of, each probe, does not require the provision of up-converting labels, and is readily automated. Time and cost efficient detection of mutant genetic sequences has been the rate limiting step in correlating mutant genotypes with altered phenotypes. Although conventional DNA sequencing methods have been considered to be the most accurate means of identifying mutations, these methods have been relatively slow and labor intensive, and are not particularly well-suited to rapidly screening large numbers of samples of genomic DNA for purposes including medical diagnosis, genomic sequencing and mapping.
All references and prior patent applications cited herein are incorporated herein by reference in their entireties.